Changes in the levels of microRNAs and effects of over-expression of microRNAs have already been related to human malignancy. However, the mechanisms by which microRNAs control cellular states and how this control is altered under stress conditions and in stationary verses proliferating cells have not been investigated. We present preliminary data indicating that microRNA-mediated regulation is important during the cell's response to stress and propose to study this relationship in normal and malignant cells. Preliminary bioinformatic analysis suggests that many mRNAs that are targets of microRNA regulation also contain conserved sites for RNA binding proteins that are known to control translation and mRNA stability during stress. mRNAs targeted by both microRNAs and stress-related RNA binding proteins can be preferentially expressed during stress. We propose to characterize the differential binding of proteins to Argonaute/microRNA complexes under stress and non-stress conditions using Stable Isotope Labeling with Amino acids in Cell culture (SILAC). We present preliminary evidence detecting differences in bound proteins under these two conditions. These proteins will be analyzed for their roles in cellular processes such as translational regulation, subcellular targeting, and mRNA stability. We further propose to identify the total set of mRNAs targeted by microRNAs by selective immunoprecipitation of Argonaute bound mRNAs from Dicer- negative embryonic stem cells, thus deficient in endogenous microRNAs that have been transfected with a single microRNA. In total, these experiments should reveal the importance of microRNA- regulation during stress conditions and could identify drug targets that could be used to preferentially inhibit/kill tumor cells undergoing stress-associated tumorigenesis. Gene regulation by microRNAs could also change during tumorigenesis if the target sites in 3'UTRs disappear. We have discovered that a large number of genes is expressed with short 3'UTRs during proliferation and longer 3'UTRs in quiescent cells. This shift was observed when arrays were used to compare mRNA expression in resting CD4-T cells and receptor stimulated CD4 cells. Further bioinformatic analysis shows this shift occurs in most resting verses proliferating tissues and in tumor verses normal tissue. The long 3'UTRs in resting cells almost certainly mediate enhanced microRNA regulation because they contain conserved seed target sites. We proposed to continue this analysis by investigating the nature of the factors controlling the proliferation-dependent shift, the importance of this change in microRNA control of the malignant phenotype, and how this shift can be modulated to induce more microRNA regulation in cancer cells. PUBLIC HEALTH RELEVANCE: The burden of cancer in public health is apparent in both human suffering and the cost of healthcare. The proposed research will provide the basis for new therapeutics to better treat cancer and thus improve public health.